We present three cases of preimplantation genetic
diagnosis (PGD) performed for hereditary cancer
syndromes at the Centre of Assisted Reproduction
and Embryology, The University of Hong Kong,
Queen Mary Hospital in Hong Kong.

Case 1

A 33-year-old woman was referred for consideration
of PGD because she was a BRCA2 gene mutation
carrier. She had cancer of the right breast at
the age of 24 years and underwent modified
radical mastectomy with axillary dissection and
immediate latissimus dorsi flap reconstruction.
Adjuvant chemoradiotherapy was given and she
was prescribed tamoxifen for 5 years after the
operation. Her paternal grandmother had breast
cancer diagnosed at the age of 60 years. Genetic
screening was performed and confirmed the patient
to be a BRCA2 mutation carrier. Her elder brother
and her father underwent the spot test and were
found to carry a BRCA2 mutation but her younger
sister was not affected. Laparoscopic ovarian
cystectomy was performed for a hyperechoic cyst
noted over the right ovary, which was confirmed to
be an endometriotic cyst. After a multidisciplinary
meeting of clinical geneticists, breast surgeons,
oncologists, gynaecologists, psychologists, nurses,
and academics in the ethics department, followed by
psychological assessment and also counselling, she
was offered in-vitro fertilisation (IVF) and PGD. Her
IVF and PGD cycle was performed in 2011, using
an antagonist protocol with letrozole co-treatment.
Fifteen oocytes were retrieved and 12 were fertilised
following intracytoplasmic sperm injection (ICSI).
Blastomere biopsy was performed on eight good-quality
cleaving embryos and five were confirmed
to be free of the BRCA2 mutation. Two unaffected
blastocysts were transferred, resulting in a singleton
pregnancy and one unaffected blastocyst was
cryopreserved. She delivered a baby boy at term by
caesarean section. Postnatal cord blood confirmed
that the baby boy did not carry the BRCA2 mutation.

Case 2

A 33-year-old woman was referred for PGD because
she was a carrier of FAP truncating germline mutation
APC c.532-8G>A (NG_008481:g93262G>A)
with a strong family history of colonic cancer. She
underwent colonoscopy surveillance and more than
100 small colonic polyps were found. She was advised
to have a prophylactic colectomy but was firm in her
request to get pregnant with PGD treatment before
the definitive treatment while fully understanding
the risks of malignancy because of the delay in
definitive treatment. She underwent an IVF cycle in
2012. Of 19 oocytes retrieved, 16 underwent ICSI.
Fifteen were fertilised and 15 embryos were available
for blastomere biopsy on day 3. Five embryos were
found without the FAP mutation and four good-quality
blastocysts were cryopreserved due to the risk
of ovarian hyperstimulation syndrome. She failed to
conceive in two frozen embryo transfer (FET) cycles
with one blastocyst replaced in each cycle. She
subsequently underwent the last FET cycle with two
blastocysts transferred, and a consequent singleton
pregnancy. The pregnancy is 26 weeks’ gestation at
the time of writing. The couple refused an invasive
prenatal test and requested postnatal cord blood
confirmation.

Case 3

A 37-year-old patient was referred from a clinical
geneticist for PGD as her husband was diagnosed to
have neurofibromatosis type I and was a carrier of
c.4495 C to T (p.Gln1499X) mutation in NF1 gene.
The mutation is a nonsense mutation that changes
the codon to a STOP codon. This mutation has
not been reported to be associated with NF1, but
such mutation is expected to result in a truncated
protein product and is therefore very likely to be
pathogenic. The couple were counselled accordingly
and were very keen for PGD treatment. The woman
underwent the first IVF cycle but only four oocytes
were retrieved. Two mature oocytes were injected
but only one was fertilised. The couple requested
cryopreservation of the only embryo on day 2. She
underwent a second IVF cycle with five oocytes
retrieved: four mature oocytes underwent ICSI
and three were fertilised. The cryopreserved day-2
embryo from the first cycle was thawed and cultured
for 24 hours. A total of four embryos were available
for embryo biopsy on day 3 and three were found
to lack the NF1 mutation. Two blastocysts were
transferred with a resulting singleton pregnancy. One
surplus blastocyst was cryopreserved. After detailed
counselling, the couple requested amniocentesis to
confirm the PGD diagnosis and amniocentesis will
be arranged at 16 to 18 weeks’ gestation (at the time
of writing the pregnancy is 14 weeks’ gestation).

The exact genomic deletion breakpoints on the
BRCA2 gene were unknown when the patient first
presented to us (the breakpoint was subsequently
studied—c.7436_7805del [GeneBank U43746])1 and
the DNA of the patient’s parents was unavailable.
Therefore we tried to establish the haplotype around
the BRCA2 gene with the sibling DNA of the non-carrier
sister and carrier brother. Nonetheless, the
patient shared no common haplotype with her non-carrier
sister and had exactly the same haplotype as
her carrier brother around the BRCA2 gene. Finally,
the high-risk haplotype was delineated by haplotype
analysis of single sperms from her carrier brother.
A PGD protocol was established that involved
whole-genome amplification,2 linkage analysis with
intragenic single-nucleotide polymorphism markers
(rs1801406 and rs1799955) and flanking micro-satellite
markers D13S289, D13S1698, D13S1701 and
D13S171, located within 2 Mb region flanking BRCA2 gene.

Case 2

Case 2 was a carrier of APC c.532-8G>A
(NG_008481:g93262G>A). The mutation was
directly determined by minisequencing with
SNaPshot Multiplex Kit (Applied Biosystems, Foster
City, US). Linkage analysis was performed by the
haplotyping method3 on a panel of 8 to 10 informative/partially informative microsatellite markers located
within a 2 Mb region flanking the APC gene. During
the pre-PGD workup, neither sibling DNA nor
offspring DNA was available to establish the high-risk
haplotype, therefore high-risk haplotype was
deduced from the genotype of embryos during
PGD treatment cycles, by correlating the result of
minisequencing and linkage analysis.

Case 3

The husband of case 3 was a carrier of mutation
NF1c.4495C>T (NM_000267). Mutation was directly
determined by minisequencing and linkage analysis
was performed on six informative/partially informative
markers located within 2 Mb region flanking NF1 gene. The
high-risk haplotype was established by single-sperm
haplotype analysis in the husband.

All PGD protocols were extensively validated
against 40 single lymphocytes (20 maternal and 20
paternal) of the corresponding couples. During the
PGD treatment cycle, all biopsied embryos resulted
in a definitive diagnosis.

Discussion

The British Society of Gastroenterology recommends
that all families with familial adenomatous polyposis
(FAP) and Lynch syndrome should be screened
in the context of a registry. A systematic review
revealed that registration and screening resulted in a
significant reduction in colorectal cancer incidence
and mortality.4 With earlier detection of these
cancer syndromes and a better surveillance system,
patient survival is improved.5 As prenatal diagnosis
is not a widely acceptable reproductive option, PGD
to reduce the chance of having offspring with the
same genetic predisposition to cancer is probably an
attractive option after detailed counselling regarding
the procedures and ethical concerns. It also avoids
the need for termination. One recent study from
the Netherlands showed that PGD is an acceptable
choice for couples with hereditary breast and ovarian
cancer (HBOC).6

In the first decade after the first published
paper on PGD, the technique was used as an
alternative to prenatal diagnosis for severe lethal
inherited diseases.7 Indications for PGD have since
been extended to adult-onset diseases such as
Huntington chorea and spinocerebellar ataxia as
well as diseases with incomplete penetrance such as
hereditary cancer syndromes. It has been challenged
ethically and the use of PGD in these indications
was controversial,8 although both ESHRE (European
Society of Human Reproduction and Embryology9)
and HFEA (Human Fertilisation & Embryology Authority; www.hfea.gov.uk) accepted these adult-onset
and multifactorial diseases as indications for
PGD.

Ovarian stimulation used for IVF treatment
may trigger a high oestradiol concentration that
may theoretically increase the risk of recurrence of
hormone receptor–positive breast cancer. The use
of letrozole to suppress the oestradiol concentration
during IVF has been successful; some large case
series have reported a comparable breast cancer
recurrence rate in those who did and did not undergo
IVF.1011 A case-control study also revealed that even
without letrozole, IVF treatment does not appear to
increase the chance of breast cancer in BRCA gene
mutation carriers.12

The use of prenatal invasive tests, such as
chorionic villus sampling and amniocentesis, to
confirm PGD results is controversial in adult-onset
diseases with incomplete penetrance with the need
for termination of pregnancy if the fetus is affected.13
In our case series, only one couple out of three
accepted the use of invasive prenatal tests and the
possibility of termination. A recent study revealed
that a proportion of couples with HBOC refused
prenatal testing even following natural conception.6
In view of the growing number of requests for
postnatal cord blood confirmation for these adult-onset
multifactorial diseases, its use in FAP families
was discussed in our PGD ethics committee. This
committee comprised reproductive medicine
subspecialists, a clinical geneticist, maternal fetal
medicine subspecialists, and laboratory in-charge.
The pros and cons of postnatal testing were discussed.
Since the risk of extra-colonic malignancies, such
as hepatoblastoma, in FAP families is about 500 to
750 times that of the general population and the
diagnosis is usually made before the age of 3 years,14
early diagnosis with postnatal confirmation of
possible incorrect PGD diagnosis in order to have
appropriate surveillance for these lethal malignancies
would be considered worthwhile. A large case
series also showed the importance of surveillance
in paediatric FAP carriers of whom a considerable
proportion with malignancies required treatment.15
The choice of using invasive prenatal procedures
or postnatal cord blood testing to confirm a PGD
diagnosis depends on discussion between the couple
and the multidisciplinary team about the variable
presentation of different syndromes.

Although PGD can be a practical and sound
reproductive option for couples with hereditary
cancer syndromes, awareness and knowledge
of this technique is lacking even in prosperous
developed countries such as the United States
where PGD treatment is readily available.1617 More
information about PGD should be available to the
general population, so that those who need this
technique have access to this option and appropriate
counselling.

Conclusion

The use of PGD is an alternative reproductive option
for hereditary cancer syndromes. With good case
selection, a multidisciplinary approach and support
for the patient and family, this can be an acceptable
option that takes account of the ethical concerns.
More information about this technique should be
provided to the general population and families with
hereditary cancer syndromes.